Method for producing cartilagetissue and implants for repairing encholndral and osteochondral defects as well as arrangement for carrying out the method

ABSTRACT

Cartilage tissue and implants comprising tissue are produced in vitro starting from cells having the ability to form an extracellular cartilage matrix. Such cells are brought into a cell space ( 1 ) and are left in this cell space for producing an extracellular cartilage matrix. The cells are brought into the cell space to have a cell density of ca. 5×10 7  to 10 9  cells per cm 3  of cell space. The cell space ( 1 ) is at least partly separated from a culture medium space ( 2 ) surrounding the cell space by means of a semi-permeable wall ( 3 ) or by an open-pore wall acting as convection barrier. The open-pore wall can be designed as a plate ( 7 ) made of a bone substitute material and constituting the bottom of the cell space ( 1 ). The cells settle on such a plate ( 7 ) and the cartilage tissue growing in the cell space ( 1 ). The cells settle on such a plate ( 7 ) and the cartilage tissue growing in the cell space ( 1 ) grows into pores or surface roughness of the plate, whereby an implant forms which consists of a bone substitute plate ( 7 ) and a cartilage layer covering the plate and whereby the two implant parts are connected to each other in positively engaged manner by being grown together.

[0001] The invention is in the field of medicinal engineering andconcerns a method according to the generic part of the first independentclaim, i.e. a method for producing cartilage tissue and implants for therepair of enchondral and osteochondral defects. Furthermore, theinvention concerns an arrangement for carrying out the method andimplants produced according to the method.

[0002] Cartilage tissue substantially consists of chondrocytes andextracellular matrix. The extracellular matrix mainly consists ofcollagen type II and proteoglycanes the components of which are exudedinto the intercellular space where they are assembled to form macromolecules. The chondrocytes make up about 5% of the volume of thecartilage tissue of a grown-up individual.

[0003] Articular cartilage coating the ends of flexibly joined bonestakes over the function of the load distribution in the loaded joint.For this function the cartilage tissue is capable to take up water andto release it again under pressure. Furthermore, the cartilage surfacesserve as sliding surfaces in the joints.

[0004] Cartilage is not vascularized and therefore its ability toregenerate is very poor, in particular in grown-up individuals and ifthe piece of cartilage to be regenerated exceeds but a small volume.However, articular cartilage often shows degenerations due to wear orage or injuries due to accidents with a far larger volume than might benaturally regenerated. This kind of defect of the cartilage layer makesmovement and strain of the affected joint painful and can lead tofurther complications such as e.g. inflammation caused by synovialliquid which comes into contact with the bone tissue due to the defectin the cartilage layer covering the bone.

[0005] For these reasons efforts have been made for quite some time toreplace or repair missing or damaged cartilage, especially articularcartilage by corresponding surgery.

[0006] It is known to repair defects concerning articular cartilage orarticular cartilage and the bone tissue beneath it by milling the defectlocation to form a bore of an as precise geometry as possible, byextracting a column of cartilage and bone of the same geometry from aless strained location of e.g. the same joint by means of boring orpunching and by inserting this column into the bore. In the same manner,larger defects with several bores are repaired (mosaic plasty). Thesemethods are successful but the actual problem is substantially shiftedfrom a strained part of a joint to a less strained part of the joint andtherefore, is not really solved.

[0007] It is also suggested, e.g. in the publication U.S. Pat. No.3,703,575 (Thiele), to repair defects of cartilage with purelyartificial implants (e.g. gels containing proteins and polysaccharides).It shows, however, that only restricted success can such be achieved andtherefore in recent development solutions to the problem have beenthought in various directions, in particular based on vital autologousor homologue cells. Vital chondrocytes or cells able to take over achondrocyte function are e.g. cultivated in vitro and then implanted; orvital chondrocytes are introduced in artificial implants; or vitalcartilage tissue is cultivated at least partly in vitro and is thenimplanted. This means that in these recent developments the aim is toproduce vital cartilage in vitro and to implant such cartilage or topopulate a defect site with cartilage forming cells which cells are thento build tissue at least similar to cartilage.

[0008] Examples of such methods are described in the followingpublications:

[0009] According to the method described in U.S. Pat. No. 4,846,835(Grande), chondrocytes taken from the patient are multiplied in amono-layer culture and, for further reproduction, are then introducedinto a three-dimensional collagen matrix in form of a gel or a sponge inwhich matrix they settle and become immobile. After ca. three weeks ofcell reproduction, the defect cartilage location is filled with thematerial consisting of the collagen matrix and the cells. In order tohold the implant in the defect location, a piece of periosteum issutured over it. The cartilage regeneration in the region of this kindof transplant is considerably better than without the transplant.

[0010] According to the method described in U.S. Pat. No. 5,053,050(Itay), chondrocytes or cells able to take over a chondrocyte functionare introduced into a biocompatible, resorbable matrix (32×10⁶ to120×10⁶ cells per cm³) in which matrix the cells are immobilized. Thismatrix is implanted, whereby a cartilage-like tissue forms in vivo. Thechondrocytes used for the implant are previously cultivated, first in amono-layer culture and then suspended, whereby they assemble to formaggregates of 30 to 60 cells.

[0011] According to the method described in U.S. Pat. No. 4,963,489(Naughton), again a three-dimensional, artificial matrix is used ascarrier material for the implant. This matrix is used for the cellculture preceding the implantation and is covered with a layer ofconnective tissue for better adhesion and better supply of the cells tobe cultivated. After in vitro cell reproduction on the three-dimensionalmatrix, the matrix is implanted. The implanted cells form the cartilagetissue in vivo.

[0012] According to the method described in PCT-WO90/12603 (Vacanti etal.), again a three-dimensional matrix is used which matrix consists ofdegradable polymer fiber materials and on which matrix the cells settle.The cells cultivated on the matrix or in mono-layer cell cultures andthen introduced into the matrix are implanted adhering to the matrix andtherefore, in an immobilized state. The matrix is degraded in vivo andis gradually replaced by extracellular matrix built by the cells.

[0013] According to the method described in U.S. Pat. No. 5,326,357(Kandel), chondrocytes are applied to a layer of filter material(MILLICELL®-CM having a pore size of 0.4 μm) in a mono-layer with a celldensity of 1.5×10⁶ cells per cm². In vitro culturing of the monolayerproduces a thin cartilage layer in two to four weeks which, in itsstructure obviously corresponds to the natural articular cartilage andcan be implanted as such.

[0014] It is also known that cartilage can be cultivated in so calledhigh density cell cultures. Cells are applied to a carrier and arecultured in a higher density than used for mono-layer culturing. Theculture medium is added only one to two hours after bringing the cellsonto the carrier. After one to three culture days, the cell layer on thecarrier contracts and so-called microspheres with diameters in the rangeof 1 mm form. On further culturing, a cartilage-like tissue forms insidethese microspheres while fibrous cartilage (perichondrium) forms ontheir surface. For implants, this kind of inhomogeneous tissue is notsuitable.

[0015] Sittinger et al. (Biomaterials Vol. 17, No. 10, May 1996,Guilford GB) suggest to introduce vital cells into a three-dimensionalmatrix for growing cartilage in vitro and to then enclose the loadedmatrix into a semi-permeable membrane. During the cartilage growth, thismembrane is to prevent the culture medium to wash away compoundsproduced by the cells and being used for constructing the extracellularmatrix. Implantation of cell cultures enclosed in this kind of membranesis also known for preventing immune reactions.

[0016] All methods named above attempt to produce cartilage at leastpartly in vitro, i.e. to produce cartilage using vital natural cellsunder artificial conditions. The problem encountered in these attemptsis the fact that chondrocytes in these in vitro conditions have thetendency to de-differentiate into fibroblasts relatively rapidly, or thefact that it is possible to differentiate fibroblasts to a chondrocytefunction under very specific culture conditions only. By thede-differentiation the chondrocytes among other things loose the abilityto produce type II collagen which is one of the most important compoundsof cartilage tissue.

[0017] According to the methods mentioned above, the problem ofde-differentiation of chondrocytes is solved by immobilizing thechondrocytes in correspondingly dense cultures in a monolayer or in athree-dimensional matrix. It shows that in this manner chondrocytesreproduce themselves without substantial de-differentiation and form anextracellular matrix which is at least similar to the extracellularmatrix of natural cartilage. The three-dimensional matrix is mostly notonly used for immobilizing the cells but also for mechanical stabilityafter implantation which is needed because none of the cartilage tissuesproduced in the named manner has a stability which could withstand evena greatly reduced strain.

[0018] The object of the invention is to create a device with whichsuitable cartilage tissue or implants which at least partly consist ofsuch cartilage tissue can be produced in vitro, the cartilage or implantserving for implantation, especially implantation in articular cartilagedefects. For achieving this object it is necessary to create an in vitroenvironment, in particular a three-dimensional such environment, inwhich environment chondrocytes or other cells capable of a chondrocytefunction do not de-differentiate over a longer culture period andperform their function actively or in which environment cellsdifferentiate to become active chondrocytes respectively. By solving theproblem of this environment, the main part of the object is achieved asnot de-differentiating, vital chondrocytes according to their naturalfunction produce the extracellular matrix characteristic for cartilageand together with this form the cartilage tissue to be created.

[0019] The implants produced according to the invention consist at leastpartly of cartilage tissue produced in vitro and are especially suitedfor the repair of enchondral or osteochondral joint defects. They are tobe producible for any possible depth of such a defect and a defectrepaired with the inventive implant is to be able to carry a normal loadas soon as possible after implantation, i.e. a load created either bypressing or by shearing forces.

[0020] This object is achieved by the method as defined in the claims.

[0021] The inventive method is based on the finding that chondrocytescan build satisfactory cartilage tissue if it is made possible that asufficiently high concentration of compounds produced by the cells andsegregated into extracellular spaces is achieved in a short initialphase and is maintained during the whole culture period. Under theseconditions the differentiated function of the chondrocytes is fullymaintained (they do not de-differentiate into fibroblasts) and/or it ispossible to differentiate corresponding cells, especially mesenchymalstem cells or other mesenchymal cells or even fibroblasts to acorresponding function.

[0022] The first condition is fulfilled by creating a cell community inwhich there is, at least at the beginning of the culture period, adensity of cells such that the cells are capable to produce the amountof compounds necessary for the mentioned concentrations in the spacesbetween the cells. The second condition is fulfilled by accommodatingthe cell community in a restricted cell space in which washing out ofthe named compounds from the extracellular spaces is prevented.

[0023] The named compounds are especially autocrine factors andsubstances serving as components for building the extracellularstructure. These components are especially aggrecanes, link-proteins andhyalorunates for building proteoglycane-aggregates and preliminarystages of collagens to eventually form collagen-fibrils of type II.

[0024] According to the inventive method, the cells are not immobilizedfor the in vitro culture but have space at their disposition, spacewithout a three-dimensional, artificial matrix in which space the twoconditions mentioned above are fulfilled, in which space it is howeverlargely left to the cells how they are to settle relative to each other.It shows that in this kind of free cell space, cells fully practicetheir chondrocyte-function and a cartilage tissue having sufficientstability for implantation and being able to carry at least part of thenormal load after implantation can be cultured.

[0025] According to the inventive method, cells which are capable of achondrocyte-function are introduced into an empty cell space, i.e. intoa space containing culture medium only, such that there is a celldensity of ca. 5×10⁷ to 10⁹ cells per cm³ in the cell space. Thisdensity amounts to a space occupation of ca. 5% to 100% at anapproximate cell volume of 10³ μm³.

[0026] The cell space has at least partly permeable walls and isintroduced into a space filled with culture medium for the length of theculture period which medium is periodically renewed in known manner.During the culture period, the cell space is arranged to be stationaryin the culture medium or it is moved in it (relative movement betweencell space and culture medium surrounding the cell space).

[0027] The permeability of the permeable parts of the cell space walland the relative movement are to be matched to the relative dimension ofthe cell space (depending on the cartilage to be produced) such that thecondition of the washing-out-prevention is fulfilled.

[0028] For all cases, semi-permeable wall regions (semi-permeablemembranes) with a permeability of 10.000 to 100.000 Dalton (10 to 100kDa) are suitable, especially for agitated cultures and for cell spacesof large dimensions (three-dimensional forms). The named autocrinefactors and components for building the macromolecules of theextracellular cartilage matrix have molecular weights which are suchthat they cannot pass through a membrane with the named permeability.

[0029] It shows that for stationary cultures and cell spaces with atleast one small dimension (thin layers) open-pore walls withconsiderably larger pores (up to the region of 10 to 20 μm) which cannotbe effective as semi-permeable walls but merely as convection barriersare sufficient and that possibly the cell space can even be open on oneside.

[0030] Especially in cell spaces not being moved and containing cells ata density in the lower region of the given density range, the cellssettle in the direction of gravity and form cartilage tissues in theform of layers. For producing more three-dimensional cartilage forms,agitated cell spaces prove to be advantageous.

[0031] For specific cultures and especially for specific forms and sizesof cell spaces the optimal arrangement (with or without movement of thecell space in the culture medium) and the optimal condition of the wallof the cell space or the permeable parts of this wall respectively mustbe determined by experiment.

[0032] The cell space has substantially three functions:

[0033] The cell space keeps the community of the (not immobilize) cellstogether at a sufficient density such that they do not loose theirspecific functionality;

[0034] the cell space restricts the growth of cartilage such that bychoice of the form of the cell space the form of the cartilage beingformed is controlled;

[0035] the cell space wall allows the supply of the cells with culturemedium but prevents the washing out of the substances produced by thecells and necessary for the growth of cartilage.

[0036] For producing implants which only partly consist of cartilagetissue, a part of the permeable wall of the cell space can additionallyhave the function of an implant part as will be described in more detailfurther below.

[0037] The cells to be brought into the cell space are chondrocytes,mesenchymal stem cells or other mesenchymal cells. These cell types areisolated in known manner from cartilage tissue, from bone or bone marrowor from connective tissue or fatty tissue. Fibroblasts are also suited,e.g. if factors are added to the culture medium or the cell space whichfactors effect differentiation of the fibroblasts to chondrocytes or ifthe cells are treated with this kind of factor before they are broughtinto the cell space. The cells can also be multiplied in vitro beforebeing brought into the cell space.

[0038] It is not necessary to isolate specific cell types from donortissue, i.e. mixtures of different cells as usually contained in suchtissues can be brought into the cell space as such. It also shows that acomplete separation of the cells from the intracellular matrix of thedonor tissue is not necessary and thus possibly tissue particles ormixtures of isolated cells and tissue particles can be brought into thecell space instead of cells only. However, care has to be taken that thenecessary cell density is achieved in the cell space possibly by partlyseparating the cells from their extracellular matrix, e.g. by means of ashort enzymatic digestion.

[0039] It shows that with the known culture media such as e.g. HAM-F12to which 5 to 15% serum is advantageously added, good results can beachieved. Furthermore, known growth factors and other components ofculture media which support the reproduction of the cells and theforming of the cartilage matrix can be added to the culture medium.

[0040] It shows that in the culture conditions created according to theinventive method the chondrocytes remain active and do notde-differentiate such that the space is filled with cartilage tissue ina culture period in the range of ca. three weeks. The cartilage tissuebeing formed can, as soon as it has a sufficient mechanical strength, beremoved from the cell space and e.g. be further cultivated floatingfreely in the culture medium or it can remain in the cell space up todirectly before being used.

[0041] Cartilage tissue produced according to the inventive method isused as implant as implant part or when containing autologous cells ascell-autotransplant or it can be used for scientific in vitro purposes.

[0042] The following Figures illustrate the inventive method, thearrangement for carrying out the inventive method as well as examples ofimplants produced by means of the inventive method. The shown examplesare implants for repair of enchondral and osteochondral defects injoints. However, using the inventive method it is also possible toproduce other implants such as e.g. auditory bones or cartilage forplastic surgery e.g. nose cartilage, orbital floors, ear conchs or partsthereof.

[0043] FIGS. 1 to 4 show four exemplified arrangements (in section) forcarrying out the inventive method for in vitro production of cartilagetissue;

[0044] FIGS. 5 to 7 show chondroitin and collagen contents of differentexperimental cartilage cultures (of cultures according to the inventivemethod and of reference cultures according to known methods) incomparison with natural articular cartilage;

[0045]FIGS. 8 and 9, for illustrating the mechanical properties ofcartilage produced according to the inventive method, show forcesstraining such cartilage (FIG. 8) and straining native cartilage (FIG.9);

[0046] FIGS. 10 to 13 show light-microscopical andelectron-microscopical micrographs of cartilage produced according tothe inventive method and of native cartilage.

[0047]FIG. 14 shows a section through an implant produced according tothe inventive method as illustrated in FIG. 2 or 3, the implantcomprising a carrier layer and a cartilage layer (boundary regionbetween the two layers in section perpendicular to the layers);

[0048]FIGS. 15 and 16 show exemplified embodiments of the inventiveimplants according to FIG. 14 in section perpendicular to the cartilagelayer;

[0049] FIGS. 17 to 20 show examples of applications of implants producedaccording to the inventive method for the repair of enchondral andosteochondral joint defects (sections perpendicular to the cartilagelayer).

[0050]FIG. 1 shows in section an exemplified arrangement for theinventive in vitro production of cartilage tissue. This arrangementsubstantially consists of a defined cell space 1 into which the cellsare introduced and which is arranged in a culture medium space 2. Atleast part of the boundary between the cell space 1 and the culturemedium space 2 is formed by a permeable wall, e.g. a semi-permeablemembrane 3. The remaining parts of the boundary separating the cellspace 1 from the culture medium space 2 are not permeable and consiste.g. of plastic components which give the cell space 1 the predeterminedform and hold the semi-permeable membrane in place.

[0051] In the shown example, an inner ring 4 and two outer snap-rings 5and 6 together with two e.g. substantially circular pieces ofsemi-permeable membrane enclose a cell space 1 in the form of a circulardisc.

[0052] The semi-permeable membrane 3 has a permeability of 10.000 to100.000 Dalton. It e.g. consists of the same material as a correspondingdialyse tube. It is obvious that in the same manner as shown in FIG. 1,cell spaces of the most various forms can be created, into which spacescells are introduced and in which spaces these cells build cartilagetissue, the cartilage tissue substantially assuming the form of the cellspace 1 or the form of the one part of cell space 1 which during theculture period faces downward in the direction of gravity.

[0053] The culture medium space 2 is a freely selectable space in whichthe culture medium is periodically exchanged in known manner. If thecell space 1 is to be moved in the culture medium space 2 the culturemedium space is e.g. a spinner bottle.

[0054] Using an arrangement according to FIG. 1, the inventive method ise.g. carried out as follows:

[0055] Cells, tissue particles or mixtures of cells and/or tissueparticles as described further above are suspended e.g. in culturemedium and are introduced into the free cell space such that the celldensity is in the range between 5×10⁷ and 10⁹ cells per cm³.

[0056] The cell space is closed and introduced into the culture mediumspace and is left there for a period of time in the range ofapproximately three weeks.

[0057] The cartilage tissue formed in the cell space is removed from thecell space and is either cultivated further swimming freely on a culturemedium or is directly used as implant or as transplant or for scientificinvestigations respectively.

[0058] In the a cell space according to FIG. 1, implants consisting ofcartilage tissue, e.g. auditory bones, nose cartilage, orbital floors orparts thereof are produced.

[0059]FIG. 2 shows a further embodiment of a cell space for carrying outthe inventive method. The cell space 1 is flat and its one side islimited by an open pore, rigid or plastically deformable plate 7 made ofa possibly biologically degradable bone substitute material, the otherside by a permeable wall, e.g. a semi-permeable membrane 3 or a morecoarsely porous wall. The cell space has a height of e.g. ca. 3 to 5 mmand any flat form and extension.

[0060] A cell space as shown in FIG. 2 is especially suited for theproduction of an implant for repair of a osteochondral defect. Theimplant comprises not only the cartilage tissue grown in the flat cellspace but also the bone substitute plate 7. This bone substitute plate 7thus has substantially two functions: during the growth of thecartilage, it serves as permeable wall for the cell space 1 and in thefinished implant, it serves as anchoring substrate for the cartilagelayer, whereby after implantation this bone substitute material iscolonized in known manner by cells immigrating from the adjacent vitalbone tissue.

[0061] In order for the bone substitute plate 7 to be able to fulfillthe second function named above care must be taken that by acorresponding arrangement of the cell space in the culture medium spacethe cells settle over the whole inner surface of the bone substituteplate 7 at least for a case in which the initial cell density is suchthat the cells have a settling tendency. Therefore, the cell space 1 ise.g., as shown in FIG. 2, arranged to be stationary with the bonesubstitute plate 7 facing downward such that the cells settle on thebone substitute plate 7 due to the effect of gravity.

[0062] Furthermore, the bone substitute plate 7 must be formed such thatthe cartilage tissue growing in the cell space 1 is able to growtogether with the bone substitute plate 7 in an intermediate region,thus forming a two part implant which can resist shearing forces. Thiskind of growing together is achieved by choosing the porosity of atleast the one surface of the bone substitute plate on which thecartilage is cultivated such that the collagen fibrils built in theextracellular cartilage matrix can grow into the pores and can suchanchor the new cartilage in the bone substitute plate. It shows that forthis kind of anchoring of the collagen fibers, pores of ca. 1 to 20 μmare suitable.

[0063] Furthermore, it is advantageous if a part of the cells which arebrought onto the surface of the bone substitute plate for cultivatingthe cartilage settle in uneven places or in pores such that the growingcartilage tissue growth is connected to the bone substitute material inthese uneven places and pores in a kind of positive engagement. It showsthat cells easily settle in uneven places or pores if these have a sizeof at least 20 μm, ideally between 20 and 50μ.

[0064] Therefore, the bone substitute plate 7 is to fulfill thefollowing conditions:

[0065] In order for the cells to be able to be nourished from theculture medium through the bone substitute plate it must comprise poreswhich form continuous canals (open porosity).

[0066] In order for the bone substitute plate to serve at least as aconvection barrier against the washing away of larger molecules thepores must not be too large and the thickness of the plate must not betoo small.

[0067] In order for the collagen fibrils being built in the growingcartilage tissue to be anchored in the pores of the plate the pores mustnot be larger than 20 μm.

[0068] In order for the cells to be able to settle in uneven places orpores in the surface of the bone substitute plate such uneven places orsurface pores (surface roughness) having sizes of at least ca. 20 μmmust be provided at least on the one part of the surface facing thegrowing cartilage layer.

[0069] It shows that with bone substitute plates having acorrespondingly rough surface, having an open porosity with pore sizesin the range of 2 to 20 μm and having a thickness of 0,5 to 3 mm,advantageously 0,5 to 1,5 mm satisfactory results can be achieved. Witha plate thickness larger than ca 0,5 to 1 mm, the region facing awayfrom the bone substitute plate can also have a coarser porosity, e.g.pores with sizes up to 300 to 700 μm such as known from bone substitutematerials. This kind of porosity favors the in vivo vascularization ofthe bone substitute material.

[0070] As bone substitute material, known osteo-inductive and/orosteo-conductive materials are suitable, advantageously biologicallydegradable such materials which have the mentioned open porosity andwhich can be processed to rigid or plastically deformable plates.Plastically deformable plates can e.g. be produced from collagen I, fromcollagen II and hydroxyapatite or from polylactic acid. Rigid plates canbe formed from tricalcium-phosphate, from hydroxyapatite or from otherinorganic bone substitute materials.

[0071] Treatment of the bone substitute plate 7 with an attachmentfactor is unnecessary.

[0072] In a cell space according to FIG. 2 comprising a correspondinglyopen-pored bone substitute plate 7, not only cartilage tissue is grownin vitro but also an implant is produced which comprises a pre-formed,grown together cartilage/bone-intermediate region.

[0073]FIG. 3 shows a further, exemplified arrangement for carrying outthe inventive method. In principle this is a combination of the methodsas carried out in arrangements according to FIGS. 1 and 2. The bonesubstitute plate 7 is not arranged as part of the permeable wall of thecell space 1 but lies within the cell space which cell space is e.g.limited by semi-permeable membranes 3. It is obvious that in this kindof arrangement the bone substitute plate 7 does not have to take overthe function of a convection barrier and that due to this the porosityand thickness of the plate can be chosen at greater liberty.

[0074] The arrangement shown in FIG. 3 is especially suitable for thin,plastically deformable bone substitute plates 7 which are complicated tohandle as cell space walls.

[0075]FIG. 4 shows schematically a further embodiment of a cell space 1which space is especially suitable for cultivating very thin cartilagelayers which are grown together with a bone substitute plate. Inopposition to the cell space according to FIGS. 1 to 3, the cell spacein FIG. 4 is open towards the top such that at least in the first one totwo weeks cultivation must take place under stationary cultureconditions. In opposition to similar, known arrangements (e.g. U.S. Pat.No. 5,326,357, Kandel) a bone substitute plate 7 is provided as carrierfor the cells and the cells are not applied to the carrier as mono-layerand are not immobilized with the help of an attachment factor.

[0076] FIGS. 5 to 7 show results from experiments with the inventivemethod (experimental arrangement substantially as outlined in FIG. 1).Dialyse tubes were used as semi-permeable membranes.

[0077] Chondrocytes were isolated from bovine shoulders using knownisolation methods. The cells were introduced into the dialyse tubes andthese were moved in a spinner bottle during the culture period, wherebythe cells settled on the bottommost end of the tubes. HAM-F12 with 5 to15% serum was used as culture medium. The culture medium was changedevery two days.

[0078]FIG. 5 shows the contents of chondroitin sulfate and collagen ofthe cartilage tissue produced according to the inventive method (in μgper ml of cartilage tissue) as a function of the duration of the cultureperiod (20, 29 and 49 days) in comparison with corresponding values ofcartilage from bovine shoulders (age: eighteen months). The results showthat the content of chondroitin sulfate can be even higher in thecartilage produced in vitro than in natural cartilage, that the contentof collagen however is considerably lower.

[0079]FIGS. 6 and 7 also show, as a function of the duration of theculture period (0, 7, 20 and 40 days), chondroitin sulfate (FIG. 6) andcollagen contents (FIG. 7) in reference cultures A and B and ofcartilage tissue grown according to the inventive method after 40 daysof culture time (C: cartilage growing in the cell space in the region ofthe settled cells, D: culture medium in the cell space above the settledcells). For references, chondrocytes were embedded in alginate spheresand cultivated in a stationary culture (reference A) and in a spinnerbottle (reference B).

[0080]FIGS. 6 and 7 make it clear that the cartilage structure in theexperimental arrangement according to the invention is considerably moresuccessful than in the reference experiments.

[0081]FIGS. 8 and 9 show results of stress experiments on cartilageproduced according to the invention (FIG. 8) and on native cartilage(FIG. 9) in order to illustrate the mechanical properties of thecartilage produced according to the inventive method (for cultureconditions see above) compared with the mechanical properties of nativebovine cartilage. The experiment consists in pressing a punch into thecartilage with a constant speed (1 micrometer per second) and to stopthe punch at a penetration depth of 200 μm while registering the punchforce. This force first rises approximately proportionally to thepenetration depth and after stopping the punch decreases (visco-elasticforce reduction due to loss of liquid of the cartilage tissue).

[0082] The two FIGS. 8 and 9 show the punch force in Newton (N) as afunction of the time in seconds (s). In FIG. 8 the registering of thedepth of penetration (travel) is shown in μm.

[0083] The stress experiment with the cartilage produced according tothe invention (FIG. 8) was carried out with a punch having a diameter of20 mm and shows a maximal pressure of ca. 0.8N/cm². The experiment withthe native cartilage was carried out with a punch having a diameter of 5mm and shows a maximal pressure of ca. 30N/cm².

[0084] The considerably smaller maximal pressure of the cartilageproduced according to the inventive method can be explained inconnection with FIGS. 10 to 13. These Figures show light-microscopicalmicrographs (FIGS. 10 and 11) and electron-microscopical micrographs(FIGS. 12 and 13) of cartilage produced according to the inventivemethod (FIGS. 10 and 11) and of human cartilage from the medium zone(FIGS. 12 and 13).

[0085]FIGS. 10 and 11 clearly show that the cartilage produced accordingto the inventive method contains considerably more chondrocytes than thenative cartilage which can be interpreted as growth stage for thecartilage cultivated in vitro. The same interpretation is suggested byFIGS. 12 and 13 in which organelles are easily visible in thechondrocytes (marked with arrows). In the native cartilage, theorganelles are narrow suggesting little synthesis activity; in thecartilage produced according to the invention they are distinctlyenlarged which suggests an intense synthesis activity, i.e. a growthstage.

[0086] Further electron-microscopical investigations of inventivelyproduced cartilage tissue show that the collagen fibrils form a densenet therein but that they are thinner than in native cartilage (aftercompleted growth) and that they are arranged having random directions.Therefore, the cartilage tissue produced according to the invention mustbe looked at as a kind of embryonic cartilage tissue which, however hasthe ability to develop in vivo (after implantation) into ‘grown-up’cartilage.

[0087]FIG. 14 schematically shows a histological section (magnificationca. 100-fold) through the intermediate region between cartilage tissueand bone substitute plate of an implant which was produced in anarrangement according to one of the FIGS. 2 to 4. The cartilage tissue10 and the bone substitute plate 7 are connected to each other in anintermediate region in the manner of positive engaging means due to thecartilage tissue having grown into uneven surface places of the bonesubstitute plate 7.

[0088]FIGS. 15 and 16 show exemplified embodiments of implants which areproduced according to methods as described in connection with FIGS. 2 to4 (section through cartilage layer 10). The implants comprise anintermediate region in which the cartilage tissue is grown together withthe bone substitute plate 7, as shown in FIG. 14.

[0089] After a culture period of a few weeks, the bone substitute plate7 with the cartilage layer 10 having grown on it is separated from theother wall components of the cell space. Before implantation, theimplant consisting of the cartilage layer 10 and the bone substituteplate 7 is reduced to the demanded size and form, if required, and/or isfixed to a further piece 12 of bone substitute material.

[0090] For processing the implant taken from the cell space, commonsurgical methods are used, such as e.g. punching, laser cutting ormilling. For enlarging the bone substitute plate, a further piece 12 ofa similar or different bone substitute material is attached on the oneside of the bone substitute plate 7 opposite to the cartilage layer 10,using a known advantageously biologically degradable cement.

[0091] After implantation, bone forming cells from the nativeenvironment migrate into the open-pore bone substitute material of thebone substitute plate 7 and of the attached piece 12 and micro-vesselsgrow into the pores of the material. As a result of this a natural bonetissue develops which gradually replaces the bone substitute material(7, 12, 13) being gradually degraded. Hereby it is to be expected thatthe cartilage cultivated in vitro is mineralized in the intermediateregion 11.

[0092] FIGS. 17 to 20 show enchondral and osteochondral defects whichare repaired with exemplified embodiments of inventive implants.

[0093]FIG. 17 shows an enchondral defect with a defined form made bydrilling or milling, i.e. a defect which lies in the native cartilagelayer 20 and does not affect the bone tissue 21 underneath the cartilagelayer 20. This kind of defect has, in the human case, a depth ofmaximally ca. 3 mm and can affect any extent of the cartilage surface. Apiece of cartilage tissue 10′ is inserted into the prepared defect whichcartilage tissue was e.g. cultivated in an arrangement according to FIG.1 and which cartilage tissue after cultivation was made to fit the formof the defect by cutting or punching, if required.

[0094] For a satisfactory fixation of the implant in the defect it issufficient at least for small implants to slightly deform the implantelastically on implantation (press fit). With larger defects the implantis fixed with known means: e.g. with a piece of periosteum which issutured or glued over the implant, with a glue being introduced betweennative cartilage and implant (e.g. fibrin glue) or by suturing theimplant to the surrounding native cartilage.

[0095]FIG. 18 shows a small osteochondral defect which has been preparedfor implantation by drilling an opening having a defined form (surfaceextension up to ca. 10 mm, depth up to ca. 3 mm in the bone tissue),i.e. a defect which does not only affect the native cartilage tissue 20but also the bone tissue 21 beneath it. This defect is repaired with animplant according to FIG. 15, which implant is fixed with one or severalpins. Two pins are shown. The one pin 22.1 is driven through the implantfrom its surface by the surgeon, the other pin 22.2 is previouslyarranged in the bone substitute plate 7 of the implant and is driveninto the bone by pressing on the surface of the implant.

[0096] Obviously, it is also possible to fix the implant in the defectaccording to FIG. 18 with other fixing means than pins.

[0097] From FIG. 18 it can be seen that in the repaired region theconsiderable shearing forces which act upon the intermediate regionbetween bone 21 and cartilage 20 when straining the joint are taken overby the intermediate region 11 where the cartilage layer grown in vitrois grown together with the bone substitute plate 7.

[0098] Instead of an implant consisting of a cartilage layer 10cultivated in vitro and a bone substitute plate 7, as shown in FIG. 18,the same defect can also be filled with a filling substance and furtherrepaired with a piece of cartilage tissue cultivated in vitro (withoutbone substitute plate), in the same way as this is shown in FIG. 17. Fortaking up the shearing forces in such a case e.g. pins 22.1 reachingright through the implant are to be provided.

[0099]FIG. 19 shows a prepared osteochondral defect having a depth of 20to 30 mm and repaired by implantation of an inventive implant accordingto FIG. 16. Shearing forces are again taken up by the region where thecartilage layer 10 and the bone substitute plate 7 are grown together.As the cement-connection 13 is positioned within the native bone 21 itis not strained by shearing and thus need not be reinforced by means ofa pin.

[0100] The repair shown in FIG. 18 can also be carried out by fillingthe lower part of the bore with bone substitute material and byimplanting an implant according to FIG. 15, possibly by means of acement layer 13.

[0101] For larger osteochondral defects a plurality of implantsaccording to FIG. 19 can be provided (mosaic plasty).

[0102]FIG. 20 shows a large and deep osteochondral defect. It is solarge that the cartilage area to be repaired can no longer beapproximated with an even area. This kind of defect can, as indicatedfurther above, be repaired with mosaic plasty. However, as the inventiveimplants are not limited regarding surface size and as the bonesubstitute plates 7 can be made of a plastically deformable material,the defect is more easily repaired according to FIG. 20 with an implantaccording to FIG. 15. For this purpose, the defect is cut out to a depthof a few millimeters into bone 21 and to a defined form, deeper regionsare filled with a plastic bone substitute material and the implant ispositioned in the defect and fixed with suitable means.

1. An implant produced by a method for producing an implant comprisingcartilage tissue, comprising the steps of: (a) providing a cell space ofa predefined form, said cell space being limited by cell space wallshaving an inside and an outside surface, wherein said cell space wallsare at least in part semi-permeable or have an open porosity suitable asa convection barrier and wherein the inside surface of said cell spacewalls does not discourage cell attachment; (b) providing cells with achondrogenic potential of 5×10⁷ to 10⁹ per cm³ of said cell space; (c)introducing a mixture consisting of said cells and a suitable culturingmedium, or consisting of tissue particles and a suitable culturingmedium into said cell space so as to form a culture medium space; (d)positioning said cell space in culturing medium, such that at least thesemi-permeable or porous part of the cell space walls are immersed insaid culturing medium; (e) maintaining a suitable culturing conditionfor a time period of sufficient duration to allow said cartilage tissueand an implant comprising cartilage tissue to grow in said cell space,wherein the implant comprises a plate made of an open-pore bonesubstitute material, the surface of which is at least partly coveredwith a cartilage layer cultivated in vitro, whereby the cartilage layeris anchored in the bone substitute material by growing into the poresand surface unevenness of the bone substitute material.
 2. The implantaccording to claim 1 , wherein the plate made of the bone substitutematerial comprises, at least in the region facing the cartilage layer,pores of a size of 1 to 20 μm and has a thickness of 0.5 to 3 mm.
 3. Theimplant according to claim 1 , wherein the plate made of bone substitutematerial is connected to a further part made of bone substitute materialby means of a cement layer.
 4. The implant according to claim 1 ,wherein the implant is rigid or plastically deformable.
 5. A method forrepairing enchondral or osteochondral joint defects, comprising thesteps of: producing an implant comprising cartilage tissue according toclaim 1 ; and implanting said implant at a site wherein said enchondralor osteochondral defects are located.
 6. A method for producing auditorybones, nose cartilage, orbital floors, ear conchs or parts thereof,comprising the steps of: producing an implant comprising cartilagetissue according to claim 1 , wherein said predefined form is in a shapeof an auditory bone, nose cartilage, orbital floor, ear conch, or partsthereof.
 7. A method for repairing enchondral or osteochondral jointdefects, comprising the steps of: producing an implant comprisingcartilage tissue according to claim 1 ; and implanting said implant at asite wherein said enchondral or osteochondral defects are located,wherein said implant repairs said enchondral or osteochondral jointdefects.